As a result of recent breakthroughs in molecular genetics it is now possible to consider introducing genes directly into somatic cells of an individual to provide a missing or needed gene product. A possible target cell for such therapy is the keratinocyte of keratinizing epithelia. Keratinocytes cultured from a suitable donor would be transformed by the introduction of a specific gene and transplanted back to the donor. If the encoded protein is synthesized and secreted at optimal levels, it may be possible to supply a therapeutically beneficial protein either systemically or locally. Furnishing a gene product locally would have unique applications in the treatment of oral and other diseases. The proposals made in this application set out to acquire information and develop capabilities that would be needed in effecting epithelial gene therapy. The experiments deal with aspects of gene transfer, gene expression, and protein secretion in cultured human keratinocytes. The specific aims are: 1. To determine the mechanism underlying enhanced expression of an exogenous gene in differentiating keratinocytes. 2. To utilize regulatory sequences from adenovirus, a virus which has evolved mechanisms for high level expression in mucosal keratinocytes, in the construction of a mucosal specific expression vector. 3. To determine if a protein which is synthesized and secreted by keratinocytes reaches the systemic circulation. 4. To examine the capacity of keratinocytes to increase production and secretion of a gene product when additional genes are introduced into the cell. 5. To develop techniques for gene transfer directly to keratinocytes in epidermis. The results of these studies, in addition to providing a basis for developing epithelial gene therapy, will foster a better understanding of gene expression in oral and epidermal tissues.